Drug resistance and dose-limiting toxicities are major obstacles in the treatment of human cancers. Improved systemic cancer therapy can be achieved by combining current anti-cancer drugs with tumor sensitizing agents. Tumor sensitivity to chemotherapy is influenced by several factors, including recognition and repair of the DNA damage caused by anti-cancer agents. Pemetrexed is an antifolate used in the treatment of human lung cancers and mesothelioma. The success of pemetrexed regimens is influenced by dose-limiting myelosuppression and the development of acquired resistance. Within tumor cells, pemetrexed causes genomic uracil incorporation through inhibition of enzymes involved in de novo thymidine biosynthesis. Base excision repair (BER), initiated by uracil DNA glycosylase (UDG), actively recognizes and removes misincorporated uracil from the genome. This proposal will utilize lung cancer cell lines address gaps in understanding of the role of UDG in pemetrexed sensitivity. Using this model we will address the hypothesis that UDG expression is induced in response to pemetrexed treatment via regulation of the nuclear UDG promoter by transcription factors involved in DNA damage response signaling (AIM1). In AIM 2, we will establish UDG-/- lung cancer cells to address the hypothesis that loss of UDG expression sensitizes lung cancer cells to pemetrexed. Lastly, in AIM3, we will use candidate UDG inhibitors to evaluate the hypothesis that inhibition of UDG activity can potentiate pemetrexed cytotoxicity. The studies proposed herein will greatly expand our knowledge of the regulation of UDG in response to DNA damage and the impact of pemetrexed- induced uracil accumulation on cancer cell toxicity, as well as evaluate the potential for UDG targeting to improve pemetrexed efficacy.